Proteins hold increasing promise for the development of new classes of analytical and therapeutic agents, but the cost of their enrichment and purification can be prohibitively high. Thus, there is a great need to develop improved processes for the purification of pharmacologically active proteins and peptides. Nature has already solved this kind of protein enrichment problem with the nuclear pore complex (NPC), the macromolecular complex that efficiently fractionates proteins between the cell nucleus and cytoplasm. Inspired by nature's solution, we have designed an artificial device that can sort nanoscale objects such as protein molecules and protein complexes and that can faithfully reproduce key features of trafficking through the NPC, including transport-factor-mediated cargo import. However, its performance still falls short of the NPC. Our goal is now to gain sufficient understanding of the molecular scale engineering principles behind nuclear transport to allow us to design next generation nanosorters capable of purifying any protein that we desire. We will first employ a "reverse engineering" approach, dissecting the architecture and operation of the NPC to determine which parameters and structures of the NPC are essential to its main transport functions, and how these central parameters have been tuned by evolution to make transport so selective and efficient in the crowded cellular environment. We will also develop methods to assess in detail the performance and mechanism of our current machines in comparison with the equivalent parameters of the NPC, to determine how they work on a molecular scale and discover why the NPC still performs better, in particular with respect to selectivity. We will then use the information garnered in these experiments to guide the design of new and improved versions of our machines, with the aim of increasing their selectivity and efficiency. Our ultimate goal is to create robust, fully synthetic, high-speed protein purification devices. We will investigate how the principles of transport by the NPC can be used to purify any protein, offering potential alternatives to chromatography, filtration and precipitation as protein purification techniques to the biotechnology industry. We will also test the use of our nanosorting devices as components of advanced, mass spectrometry-based, proteomic analytical instruments. PUBLIC HEALTH RELEVANCE: Almost all the active products from the biotechnology industry are proteins and peptides, but their purification remains a costly challenge. However, nature has already devised a rapid and efficient way to purify proteins;nuclear pore complexes sort many hundreds of proteins per second between the nucleus and cytoplasm. By discovering how nuclear pore complexes sort proteins, we are learning how to build prototype nanosorting machines, which eventually should be able to purify many kinds of medically relevant proteins and peptides far more efficiently and at far lower cost than currently possible.